Description: (from applicant's abstract) It is the aim of this grant to understand defects in vesicular trafficking and cytoskeleton that may underlie Huntington's disease. Expansion of a trinucleotide repeat CAG, encoding glutamine, results in at least eight progressive neurodegenerative disorders, including Huntington's disease (HD). The mechanism by which polyglutamine expansion selectively kills neurons is largely unknown. Aggregation is generally accepted as part of pathogenesis, but it is not known whether toxicity initiates in the nucleus or the cytoplasm. Using time lapse imaging, we have tracked the localization of huntingtin in individual neurons from expression of the mutant protein to cell death. Toxicity initiates in the cytoplasm of primary neurons. Further, we have identified targets of huntingtin-mediated aggregation directly from aggregates in human brain. We show that the expanded Huntington's protein sequesters tubulin and vesicular trafficking motors into insoluble complexes. Direct video imaging of vesicles indicates that the mutant protein indeed inhibits vesicular transport particularly in the anterograde direction. The motor that is most affected appears to be kinesin and the cargo that appears most affected is mitochondria. Our data support a model for HD pathogenesis in which aggregation inhibits proteolysis of the HD protein, disrupts cytoskeletal architecture and impairs motors required for vesicular/organelle trafficking. In this proposal, we aim to test the hypothesis that sequestration of tubulin-dependent motors underlies HD regional pathology. Using gel filtration, immunoblotting and immunoprecipitation reactions, we will evaluate whether sequestration of tubulin-dependent complexes underlies HD regional pathology in human brain. By establishing primary cultures of affected and resistant neurons, we will directly test whether vesicular trafficking is altered in the presence of normal and expanded HD protein. Since mitochondria are reduced in number in the presence of the mutant huntingtin, we will monitor the fate and subcellular localization of mitochondria by confocal imaging. Alteration in transport will be correlated with the subcellular localization of the HD protein and trafficking motors using fluorescence-labeled proteins and confocal microscopy. Finally, we will refine our understanding of pathogenesis by identifying other proteins present in aggregates by mass spectrometry. The recent observations that tubulin-dependent complexes and vesicular transport may play a role in pathogenesis of ALS and Alzheimer's disease suggest that there may be common aspects to these neurological diseases.